WO2018124766A9 - Récepteur antigénique chimérique et cellules tueuses naturelles exprimant celui-ci - Google Patents

Récepteur antigénique chimérique et cellules tueuses naturelles exprimant celui-ci Download PDF

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WO2018124766A9
WO2018124766A9 PCT/KR2017/015635 KR2017015635W WO2018124766A9 WO 2018124766 A9 WO2018124766 A9 WO 2018124766A9 KR 2017015635 W KR2017015635 W KR 2017015635W WO 2018124766 A9 WO2018124766 A9 WO 2018124766A9
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domain
car
intracellular signaling
nucleotides
nkg2d
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WO2018124766A2 (fr
WO2018124766A3 (fr
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황유경
조성유
원성용
임호용
허정현
정미영
김현아
권수현
이은솔
김한솔
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주식회사 녹십자랩셀
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Priority to CN201780081622.3A priority Critical patent/CN110121505B/zh
Priority to CA3061898A priority patent/CA3061898A1/fr
Priority to JP2019536074A priority patent/JP6971319B2/ja
Priority to US16/474,426 priority patent/US20190336533A1/en
Priority to EP17886739.6A priority patent/EP3567049A4/fr
Priority to AU2017384900A priority patent/AU2017384900B2/en
Publication of WO2018124766A2 publication Critical patent/WO2018124766A2/fr
Publication of WO2018124766A3 publication Critical patent/WO2018124766A3/fr
Publication of WO2018124766A9 publication Critical patent/WO2018124766A9/fr
Priority to US17/845,793 priority patent/US20230025506A1/en

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    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to chimeric antigen receptors and natural killer cells expressing them.
  • NK cells human killer cells
  • adoptive immune therapy adoptive cellular immunotherapy
  • Chimeric antigen receptors are known to be able to reprogram T cells to activate to increase the therapeutic effect on specific cancers or to overcome the resistance of cancer cells to treatment.
  • the OX40 ligand (CD252), a protein belonging to the TNFR superfamily, is known to be expressed in antigen-presenting cells (APCs), some natural killer cells and some B cells. It is known to be expressed within a few days.
  • OX40 (CD134), a receptor for the OX40 ligand (CD252), is known to be expressed in T cells and is known to be expressed 24 hours after activation of T cells by antigen and CD28.
  • CD134 is known to further enhance T cell response by CD28 activation, thereby enhancing T cell proliferation, cytokine secretion and survival.
  • OX40 ligand in the increase of anticancer activity of natural killer cells is not well known, and no attempt to use OX40 ligand in chimeric antigen receptor has been reported.
  • the present inventors constructed two different natural NK receptors containing CAR to investigate the functional role of CD252 in the antitumor response of NK cells.
  • lentivirus encoding the recombinant high affinity FCRG3A V158 variant (CD16V) in which various complementary stimulatory molecules known to increase the cytotoxicity of NK cells were genetically fused to the signal transduction domain.
  • CD16V receptors containing various intracellular signaling molecules were transduced into NK cells and their expression was detected and their antitumor response in the presence of rituximab specific for CD20 molecules using lymphoma cell lines in vitro Respectively.
  • NKG2D is a major receptor for NK cell activation and binds to MHC class 1 chain-related A (MICA), MICB, and various UL-16 binding proteins (ULBP) that are preferentially expressed after cell stress, infection or cytotoxicity.
  • MICA MHC class 1 chain-related A
  • MICB MHC class 1 chain-related A
  • ULBP UL-16 binding proteins
  • NK92 cells were constructed and expressed in NK92MI cells containing various signaling molecules known to induce cytotoxicity in NK cells. And then their antitumor effects were evaluated in vitro.
  • a chimeric antigen receptor containing an OX40 ligand can be usefully used as a means for overcoming the limitations of autoimmune cell therapy using natural killer cells, and it is possible to use an OX40 ligand
  • the chimeric antigen receptor is remarkably superior in the effect of increasing the anticancer activity of natural killer cells, thereby completing the present invention.
  • a chimeric antigen receptor comprising an intracellular signaling domain comprising all or part of an OX40 ligand (CD252).
  • transmembrane domain as in 1 above, wherein said transmembrane domain is linked to said intracellular signaling domain; A spacer domain connected to the membrane through domain; And an extracellular domain linked to the spacer domain. ≪ Desc / Clms Page number 13 >
  • the chimeric antigen receptor of claim 2 further comprising a signal sequence linked to said extracellular domain.
  • Fv fragment is a single-chain variable fragment (ScFv).
  • chimeric antigen receptor of claim 5 wherein said natural cytotoxicity receptor is selected from the group consisting of NKp46, NKp30, NKp44, NKp80 and NKp65 receptors.
  • transmembrane domain comprises all or part of any one selected from the group consisting of CD8? And CD28.
  • a first intracellular signaling domain comprising all or part of any one selected from the group consisting of chimeric antigen receptors: CD28 and 4-1BB comprising the following intracellular signaling domains: A second intracellular signaling domain comprising all or part of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB; And a third intracellular signaling domain comprising all or part of the CD3 ⁇ , wherein said first, second and third intracellular signaling domains are located in order intracellularly from the cell membrane.
  • transmembrane domain is linked to the intracellular signaling domain; A spacer domain connected to the membrane through domain; And an extracellular domain linked to the spacer domain. ≪ Desc / Clms Page number 13 >
  • transmembrane domain comprises all or part of any one selected from the group consisting of CD8? And CD28.
  • said first intracellular signaling domain comprises all or part of CD28;
  • Said second intracellular signaling domain comprises all or part of an OX40 ligand;
  • said third intracellular signaling domain comprises all or part of CD3 <
  • RTI ID 0.0 > zeta. ≪ / RTI >
  • a pharmaceutical composition for the treatment of tumors comprising the above 23 immune cells as an active ingredient.
  • nucleic acid sequence according to 27 above wherein said nucleic acid sequence is at least one of SEQ ID NOS: 33, 41, 43, 45, 47, 49, 51, 53, 55, 69, 71, 77, 81, 83, 85, 87, ≪ / RTI > or a variant thereof having at least 80% sequence identity.
  • nucleic acid sequence is selected from the group consisting of SEQ ID NOS: 32, 40, 42, 44, 46, 48, 50, 52, 54, 68, 70, 76, 80, 82, 84, 86, ≪ / RTI > or a variant thereof having at least 80% sequence identity.
  • a method of treating a tumor comprising administering to a subject an immune cell of the above 23.
  • the chimeric antigen receptor according to the present invention has an excellent natural killer cell activation efficiency.
  • the chimeric antigen receptor according to the present invention can be used as various cancer target antibodies depending on the type of cancer to be targeted.
  • the chimeric antigen receptor according to the present invention can be applied to various carcinomas by applying various antigen recognition sites.
  • the natural killer cells expressing the chimeric antigen receptor according to the present invention are excellent in cytotoxicity against cancer cells.
  • the natural killer cells expressing the chimeric antigen receptor according to the present invention can be useful for the treatment of immune cells.
  • FIG. 1A is a block diagram of a CD16V-ZZ CAR (first generation), a CD16V-28Z CAR (second generation), a CD16V-BBZ CAR (second generation), a CD16V-OX40Z CAR The expression level of each CAR in NK92MI cells transduced with CAR (3rd generation) is shown.
  • FIG. 1B is a block diagram of a CD16V-ZZ CAR (first generation), a CD16V-28Z CAR (second generation), a CD16V-BBZ CAR (second generation), a CD16V-OX40Z CAR
  • the intrinsic (instrinsic) cell killing ability of NK92MI cells transduced with CAR (3rd generation) is shown through a cytotoxicity assay on K562 cells.
  • FIG. 1C is a block diagram of a CD16V-ZO CAR (first generation), a CD16V-28Z CAR (second generation), a CD16V-BBZ CAR (second generation), a CD16V- This shows the evaluation of the killing ability of natural killer cells in combination with antibodies to Ramos cells of NK92MI cells transfected with CAR (3rd generation).
  • FIG. 1D shows the expression levels of each CAR in NK92MI cells transduced with CD16V-Z CAR (first generation) and CD16V-OX40LZ CAR (second generation) according to an embodiment of the present invention.
  • FIG. 1E shows the evaluation of the killing ability of natural killer cells in combination with antibodies against Ramos cells of NK92MI cells transduced with CD16V-Z CAR (first generation) and CD16V-OX40LZ CAR (second generation) according to an embodiment of the present invention .
  • Figure 1F shows the expression levels of each CAR in NK92MI cells transduced with CD16V-Z CAR (first generation) and CD16V-ZOX40L CAR (second generation) according to an embodiment of the present invention.
  • FIG. 1G shows the evaluation of the killing efficiency of natural killer cells in combination with antibodies against Ramos cells of NK92MI cells transfected with CD16V-Z CAR (first generation) and CD16V-ZOX40L CAR (second generation) according to an embodiment of the present invention .
  • FIG. 2A shows the expression of each CAR in NK92MI cells transduced with CD16V-Z CAR (first generation), CD16V-BBZ CAR (second generation), or CD16V-BBOX40LZ CAR (third generation) according to an embodiment of the present invention .
  • Figure 2B is a graph showing the effect of NK92MI cells expressing CD16V-Z CAR (first generation), CD16V-BBZ CAR (second generation), or CD16V-BBOX40LZ CAR (third generation) Of the natural killer cells.
  • Figure 3A shows the expression levels of each CAR in NK92MI cells transduced with CD16V-28OX40LZ CAR, CD16V-28OX40Z CAR, or CD16V-28BBZ CAR, third generation CARs according to an embodiment of the present invention.
  • FIG. 3B is a graph showing the results of evaluating the killing efficiency of natural killer cells in combination with antibodies against Ramos cells of NK92MI cells expressing CD16V-28OX40LZ CAR, CD16V-28OX40Z CAR, or CD16V-28BBZ CAR, which are third generation CARs according to the embodiment of the present invention .
  • FIG. 4B is a graph showing the results of a comparison of CD16V-28 (H) BBZ CAR, CD16V-28 (H) OX40Z CAR, or CD16V-28 (H) OX40LZ CAR, third generation CARs containing CD28 in a hinge according to an embodiment of the present invention. This shows the evaluation of the killing ability of natural killer cells in combination with antibodies against Ramos cells of NK92MI cells.
  • FIG. 6B is a graph showing the effect of the NKG2D-28BBZ CAR (third generation) and the NKG2D-28OX40Z CAR (third generation) transfected with NKG2D-Z CAR (first generation), CD28 signaling domain and NKG2D-28BBZ CAR This shows the evaluation of the killing ability of natural killer cells against the human breast cancer cell line MCF-7.
  • FIG. 7A is a graph showing the relationship between the NKG2D-Z CAR (first generation), NKG2D-28Z CAR (second generation), NKG2D-28 (H) OX40LZ CAR (third generation), NKG2D extracellular domain and CD28 hinge AAA-28 (H) OX40LZ CAR (3rd generation) containing the AAA sequence in the NK92MI cells transfected with the NK92D cells.
  • FIG. 7B is a graph showing the relationship between the NKG2D-Z CAR (first generation), NKG2D-28Z CAR (second generation), NKG2D-28 (H) OX40LZ CAR (third generation), NKG2D extracellular domain and CD28 hinge 6 shows the evaluation of the killing ability of NK92MI cells transfected with NKG2D-AAA-28 (H) OX40LZ CAR (3rd generation) containing the AAA sequence against the human breast cancer cell line MCF-7.
  • Figure 8B shows the expression levels of various NKG2D ligands in human lung cancer cell lines H1299 and H1944 according to an embodiment of the present invention.
  • the present invention relates to a chimeric antigen receptor and a natural killer cell expressing the chimeric antigen receptor, and includes an intracellular signaling domain including all or a part of an OX40 ligand (CD252), thereby enhancing the anticancer activity of immune cells Chimeric antigen receptor (CAR) and immune cells expressing the chimeric antigen receptor.
  • CD252 an OX40 ligand
  • the chimeric antigen receptor of the invention comprises an intracellular signaling domain comprising all or part of an OX40 ligand (CD252).
  • the chimeric antigen receptor comprises a transmembrane domain connected to the intracellular signaling domain; A spacer domain connected to the membrane through domain; And an extracellular domain connected to the spacer domain.
  • the chimeric antigen receptor may further comprise a signal sequence connected to an end of the extracellular domain not connected to the spacer domain.
  • each of the above domains may be directly connected to each other or may be connected by a linker.
  • the signal sequence may be such that when the chimeric antigen receptor is expressed, the extracellular domain can be located outside the cell membrane of immune cells (e. G., Natural killer cells).
  • the signal sequence may comprise all or part of CD16.
  • the extracellular domain is a domain that specifically binds to an antibody or specifically recognizes an antigen, and includes, for example, an Fc receptor, a single-chain variable fragment , ScFv), a natural cytotoxicity receptor, NKG2D, 2B4 or DNAM-1.
  • Fc receptor a single-chain variable fragment
  • ScFv single-chain variable fragment
  • NKG2D a single-chain variable fragment
  • DNAM-1 a natural cytotoxicity receptor
  • the chimeric antigen receptor according to an embodiment of the present invention may include an Fc receptor as an extracellular domain and thus can be used with various antibodies depending on the type of cancer cell to be treated.
  • the Fc receptor may be any one selected from the group consisting of CD16, CD32, CD64, CD23 and CD89.
  • the Fc receptor may comprise all or part of the CD16 V158 variant (CD16V).
  • the chimeric antigen receptor of the present invention may comprise an antigen-binding fragment of an antibody that recognizes a direct antigen as an extracellular domain as an extracellular domain without administration in combination with an antibody.
  • the antigen binding fragment may be an Fab fragment, F (ab ') 2 fragment, F (ab') 2 fragment or Fv fragment.
  • the antibody may be any one of various types of antibodies capable of antigen-specific binding.
  • the antibody may be a combination of one light chain and one heavy chain, and may be a combination of two light chains and two heavy chains.
  • the first unit having the first light chain and the first heavy chain bonded together and the second unit having the second light chain and the second heavy chain bonded to each other may be combined.
  • the bond may be a disulfide bond, but is not limited thereto.
  • the two unit pieces may be the same or different from each other.
  • the first monomer unit including the first light chain and the first heavy chain and the second monomer unit including the second light chain and the second heavy chain may be the same or different from each other.
  • Antibodies prepared so that the first unit and the second unit recognize two different antigens are generally referred to in the art as bispecific antibodies. Further, for example, the antibody may be a combination of three or more of the above units.
  • the antigen-binding fragments of the present invention may be derived from various types of antibodies as described above, but are not limited thereto.
  • the extracellular domain used in the present invention may be a natural cytotoxicity receptor.
  • the natural killer receptor includes, but is not limited to, NKp46, NKp30, NKp44, NKp80 and NKp65 receptors.
  • the transmembrane domain is located through the cell membrane, and any substance that can be located through the cell membrane without interfering with the function of the extracellular domain and the intracellular signal transduction domain All available.
  • the transmembrane domain may include all or a part of any one selected from the group consisting of CD8? And CD28.
  • the extracellular domain and the transmembrane domain may be connected to a spacer domain.
  • the spacer domain may be a hinge domain.
  • the spacer domain may include all or a part of any one selected from the group consisting of CD8? And CD28.
  • the intracellular signal transduction domain is located inside the cell membrane of the natural killer cell, i.e., in the cytoplasm.
  • the antibody bound to the extracellular domain of the present invention binds to the target antigen, Or a sequence capable of signaling activation of natural killer cells.
  • the chimeric antigen receptor may be one comprising one or more intracellular signaling domains. Where two or more intracellular signaling domains are involved, intracellular signaling domains may be connected in series with each other. For example, in the case of including three intracellular signaling domains, one end of a first intracellular signaling domain is connected to a terminal of the transmembrane domain that is not connected to the spacer domain, and the first intracellular signaling domain Wherein one end of the second intracellular signaling domain is connected to an end of the second intracellular signaling domain that is not connected to the membrane intracellular signaling domain of the second intracellular signaling domain, One end of the intracellular signaling domain may be connected.
  • the first, second, and third intracellular signal transduction domains may be sequentially positioned in the cell membrane in the intracellular direction. Even when the intracellular signaling domains are two, four, or more, they can be connected to each other in the same manner. According to an embodiment of the present invention, each of the above domains may be directly connected to each other or may be connected by a linker.
  • the chimeric antigen receptor may comprise two intracellular signaling domains.
  • it may include a first intracellular signaling domain connected to the transmembrane domain and a second intracellular signaling domain connected to an end of the first intracellular signaling domain that is not connected to the transmembrane domain.
  • the first intracellular signaling domain is selected from the group consisting of OX40 (CD134), OX40 ligand (OX40L, CD252), 4-1BB (CD137), CD28, DAP10, CD3-
  • the second intracellular signaling domain may include all or a part of any one selected from the group consisting of OX40 ligand, CD3-zeta, and DAP12.
  • the first intracellular signaling domain and the second intracellular signaling domain includes all or a part of the OX40 ligand.
  • the chimeric antigen receptor may comprise a first intracellular signaling domain comprising all or part of the OX40 ligand and a second intracellular signaling pathway comprising all or part of any one selected from the group consisting of CD3-zeta and DAP12 Domain.
  • the chimeric antigen receptor may comprise a first intracellular signaling domain comprising all or part of any one selected from the group consisting of CD3-zeta and DAP12 and a second intracellular signal comprising all or part of the OX40 ligand And may include a delivery domain.
  • the chimeric antigen receptor may comprise three intracellular signaling domains. For example, a first intracellular signaling domain linked to the transmembrane domain; A second intracellular signaling domain linked to an end of the first intracellular signaling domain that is not linked to the transmembrane domain of the first intracellular signaling domain; And a third intracellular signal transduction domain linked to an end of the second intracellular signal transduction domain that is not connected to the first intracellular signal transduction domain.
  • the first intracellular signal transduction domain may include all or a part of any one selected from the group consisting of 4-1BB, OX40, OX40 ligand, CD28 and DAP10
  • 2 intracellular signaling domain may comprise all or part of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB
  • the third intracellular signal transduction domain may include all or a part of any one selected from the group consisting of OX40 ligand, CD3-zeta and DAP12.
  • at least one of the first intracellular signaling domain, the second intracellular signaling domain and the third intracellular signaling domain includes all or a part of the OX40 ligand.
  • the present invention also relates to a first intracellular signaling domain comprising all or part of any one selected from the group consisting of CD28 and 4-1BB; A second intracellular signaling domain comprising all or part of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB; And a third intracellular signaling domain comprising all or part of CD3-zeta, wherein said first, second and third intracellular signaling domains are selected from the group consisting of chimeric antigen receptors Lt; / RTI > According to an embodiment of the present invention, each of the above domains may be directly connected to each other or may be connected by a linker.
  • the chimeric antigen receptor comprises a transmembrane domain connected to the first intracellular signaling domain; A spacer domain connected to the membrane through domain; And an extracellular domain connected to the spacer domain.
  • the chimeric antigen receptor may further comprise a signal sequence linked to the extracellular domain.
  • each of the above domains may be directly connected to each other or may be connected by a linker.
  • the extracellular domain is a domain that specifically binds to an antibody or specifically recognizes an antigen, and includes, for example, an Fc receptor, a single-chain variable fragment , ScFv), a natural cytotoxicity receptor, NKG2D, 2B4 or DNAM-1.
  • Fc receptor a single-chain variable fragment
  • ScFv single-chain variable fragment
  • NKG2D a single-chain variable fragment
  • DNAM-1 a natural cytotoxicity receptor
  • the chimeric antigen receptor according to an embodiment of the present invention may include an Fc receptor as an extracellular domain and thus can be used with various antibodies depending on the type of cancer cell to be treated.
  • the Fc receptor may be any one selected from the group consisting of CD16, CD32, CD64, CD23, CD89 and mutants thereof.
  • the Fc receptor may comprise CD16 or a variant thereof, and most specifically may comprise all or part of the CD16 V158 variant (CD16V).
  • the chimeric antigen receptor of the present invention may comprise an antigen-binding fragment of an antibody that recognizes a direct antigen as an extracellular domain as an extracellular domain without administration in combination with an antibody.
  • the antigen binding fragment may be an Fab fragment, F (ab ') 2 fragment, F (ab') 2 fragment or Fv fragment.
  • the antibody may be any one of various types of antibodies capable of antigen-specific binding.
  • the antibody may be a combination of one light chain and one heavy chain, and may be a combination of two light chains and two heavy chains.
  • the first unit having the first light chain and the first heavy chain bonded together and the second unit having the second light chain and the second heavy chain bonded to each other may be combined.
  • the bond may be a disulfide bond, but is not limited thereto.
  • the two unit pieces may be the same or different from each other.
  • the first monomer unit including the first light chain and the first heavy chain and the second monomer unit including the second light chain and the second heavy chain may be the same or different from each other.
  • Antibodies prepared so that the first unit and the second unit recognize two different antigens are generally referred to in the art as bispecific antibodies. Further, for example, the antibody may be a combination of three or more of the above units.
  • the antigen-binding fragments of the present invention may be derived from various types of antibodies as described above, but are not limited thereto.
  • the extracellular domain used in the present invention may be a natural cytotoxicity receptor.
  • the natural killer receptor includes, but is not limited to, NKp46, NKp30, NKp44, NKp80 and NKp65 receptors.
  • the signal sequence may comprise all or part of CD16.
  • the extracellular domain may comprise all or part of the CD16 V158 variant (CD16V).
  • the spacer domain may include all or a part of any one selected from the group consisting of CD8 alpha (CD8 alpha) and CD28.
  • the transmembrane domain may comprise all or part of any one selected from the group consisting of CD8a and CD28.
  • the chimeric antigen receptor is selected from the group consisting of SEQ ID NOS: 33, 41, 43, 45, 47, 49, 51, 53, 55, 69, 71, 77, 81, 83, 85, 87, 89, ≪ / RTI > or a variant thereof having at least 80% sequence identity.
  • the present invention also provides immune cells expressing a chimeric antigen receptor according to the present invention (for example, NK cells).
  • the immune cells of the present invention may be toxic to tumor cells. Since the chimeric antigen receptor according to the present invention determines the specific toxicity of certain tumor cells depending on which antibody binds with an extracellular domain, the immune cells expressing the chimeric antigen receptor according to the present invention are toxic.
  • the tumor cells that can be shown are not particularly limited. According to one embodiment, when the immune cells of the invention (e. G. Natural killer cells) are used with rituximab, they may exhibit toxicity to malignant lymphoma cells.
  • the malignant lymphoma cell may be one that expresses CD20.
  • the malignant lymphoma may be a B-cell lymphoma.
  • the present invention also relates to a method for producing a chimeric antigen receptor comprising the step of culturing a chimeric antigen receptor according to the present invention, wherein the number of immune cells expressing the chimeric antigen receptor according to the present invention (for example, natural killer cells) is 2 to 7.5 times as many as the number of tumor cells (e.g., malignant lymphoma cells)
  • the number of immune cells expressing the chimeric antigen receptor according to the present invention for example, natural killer cells
  • tumor cells e.g., malignant lymphoma cells
  • the pharmaceutical composition of the present invention is characterized in that the number of said immune cells (e. G., Natural killer cells) in a single dose is 0.75 times to the number of said tumor cells (for example, malignant lymphoma cells) It may be included 10 times.
  • the number of said immune cells (for example, natural killer cells) in a single dose may be 2 to 7.5 times the number of said tumor cells (for example, malignant lymphoma cells) in a subject to be treated.
  • the present invention also provides a nucleic acid sequence encoding a chimeric antigen receptor according to the present invention as described above.
  • the nucleic acid sequence is selected from the group consisting of SEQ ID NOS: 32, 40, 42, 44, 46, 48, 50, 52, 54, 68, 70, 76, 80, 82, 84, 86, 92, or a variant thereof having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:
  • the present invention also provides a vector comprising the nucleic acid sequence according to the present invention as described above.
  • the present invention also provides a method for treating a tumor, comprising the step of administering the above-described immune cells to a subject.
  • the present invention also provides a method for preventing tumor metastasis comprising the step of administering the above-described immune cells to a subject.
  • the subject may be a mammal having a tumor, and in particular may be a human, but is not limited thereto.
  • Administration can result in an amount of the immune cells expressing the chimeric antigen receptor (e. G., Natural killer cells) according to the present invention in an amount of 2 to 7.5 times the number of tumor cells (e. G., Malignant lymphoma cells) in the subject to be treated.
  • the immune cells expressing the chimeric antigen receptor e. G., Natural killer cells
  • tumor cells e. G., Malignant lymphoma cells
  • the method of administration is not particularly limited and may be administered through conventional oral or parenteral routes.
  • the tumor is not particularly limited, and may be, for example, malignant lymphoma, leukemia, breast cancer, lung cancer, and more specifically, B-cell lymphoma.
  • Example 1 Methods and reagents
  • Human breast cancer cell line H1299 and H1944 cell line and NK-92MI were supplied from ATCC (American Type Culture Collection, Manassas, VA, USA), human breast cancer cell line Ramos, human erythroleukemic cell line K562, human breast cancer cell line MCF- Respectively.
  • K562 was maintained in RPMI-1640 (Gibco, Grand Island, NY, USA) containing 10% FBS.
  • Ramos was maintained in RPMI-1640 (ATCC) (Manassas, Va.) Containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA).
  • MCF-7 was maintained in EMEM (ATCC) + 10% FBS (Gibco) medium and H1299 and H1944 cell lines were maintained in RPMI-1640 (ATCC) + 10% FBS (Gibco).
  • NK-92MI and transformed NK-92MI cells were maintained in CellGro® serum-free medium containing 1% human plasma.
  • 293T cell line, human embryonic kidney fibroblast, was supplied from ATCC and used in DMEM (Gibco, Grand Island, NY, USA) containing 10% FBS (Gibco, Grand Island, Respectively.
  • the signal sequence and extracellular domain of the FCRG3A V158 mutation (CD16V); NKG2D extracellular domain; The signal sequence of CD8 alpha, the hinge and transmembrane domain of CD8 alpha; The hinge and transmembrane domain of CD28; The intracellular signaling domains of 4-1BB, OX40, OX40 ligand (OX40L) and CD3 ⁇ were each artificially synthesized. They were assemble in various combinations using SOE-PCR (splicing by overlapping extension by PCR). PCR results were confirmed by direct sequencing.
  • PCR products were digested with Nhe1 and EcoRI and then inserted into the Nhe1 and EcoRI sites of the MSCV-EF1 ⁇ -GFP vector or EF1a-MCS vector, a third generation self-inactivating lentiviral expression vector ligation).
  • Table 1 summarizes the chimeric antigen receptor (CAR) according to the embodiment of the present invention.
  • the domains of all CARs according to an embodiment of the present invention are connected in tandem to each other and also connected in frame.
  • CD16V-Z CAR (Generation 1) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No. X52645); Hinge and transmembrane domains from human CD8 alpha (1292-1507 nucleotides, GenBank NM 001768.6); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • CD16V-BBZ CAR (2nd generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • CD16V-OX40Z CAR (second generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • CD16V-OX40LZ CAR (second generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • CD16V-ZOX40L CAR (2nd generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • CD16V-28Z CAR (Generation 2) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No.
  • CD16V FCRG3A V158
  • FCRG3A V158 The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No. X52645); Hinge from human CD8 alpha (1292-1435 nucleotides, GenBank NM 001768.6); The transmembrane and intracellular signaling domains from CD28 (679-882 nucleotides, GenBank NM 006139.3); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • CD16V-28 (H) Z CAR (2nd generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No. X52645); CD28 derived hinge, transmembrane and intracellular signaling domains (562-882 nucleotides, GenBank NM 006139.3); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • CD16V-BBOX40Z CAR (3rd generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • X52645 Hinge and transmembrane domains from human CD8 alpha (1292-1507 nucleotides, GenBank NM 001768.6); An intracellular signaling domain from CD137 (901-1026 nucleotides, GenBank NM 001561.5); An intracellular signaling domain from CD134 (733-840 nucleotides, GenBank AB590584.1); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • CD16V-OX40BBZ CAR (Generation 3) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • X52645 Hinge and transmembrane domains from human CD8 alpha (1292-1507 nucleotides, GenBank NM 001768.6); An intracellular signaling domain from CD134 (733-840 nucleotides, GenBank AB590584.1); An intracellular signaling domain from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • CD16V-28BBZ CAR (3rd generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • CD16V-28OX40Z CAR (3rd generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • CD8 alpha (1292-1435 nucleotides, GenBank NM 001768.6)
  • the transmembrane and intracellular signaling domains from CD28 (679-882 nucleotides, GenBank NM 006139.3);
  • An intracellular signaling domain from CD134 (733-840 nucleotides, GenBank AB590584.1);
  • an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • CD16V-28OX40LZ CAR (3rd generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • CD16V-28 (H) BBZ CAR (3rd generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • CD28 derived hinge, transmembrane and intracellular signaling domains (562-882 nucleotides, GenBank NM 006139.3); An intracellular signaling domain from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • CD16V-28 (H) OX40Z CAR (3rd generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • CD28 derived hinge, transmembrane and intracellular signaling domains (562-882 nucleotides, GenBank NM 006139.3); An intracellular signaling domain from CD134 (733-840 nucleotides, GenBank AB590584.1); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • CD16V-28 (H) OX40LZ CAR (3rd generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • CD28 derived hinge, transmembrane and intracellular signaling domains (562-882 nucleotides, GenBank NM 006139.3); An intracellular signaling domain from CD252 (141-206 nucleotides, GenBank NM 003326.4); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • CD16V-BBOX40LZ (third generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); The extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of nucleotide 559 in GenBank Accession No.
  • X52645 Hinge and transmembrane domains from human CD8 alpha (1292-1507 nucleotides, GenBank NM 001768.6); An intracellular signaling domain from CD137 (901-1026 nucleotides, GenBank NM 001561.5); An intracellular signaling domain from CD252 (141-206 nucleotides, GenBank NM 003326.4); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • CAR chimeric antigen receptor
  • Table 3 summarizes the chimeric antigen receptor (CAR) according to the present invention.
  • the domains of all CARs according to an embodiment of the present invention are connected in tandem to each other and also connected in frame.
  • NKG2D-Z CAR (Generation 1) is the signal sequence domain of CD8 alpha (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains from human CD8 alpha (1292-1507 nucleotides, GenBank NM 001768.6); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-BBZ CAR (second generation) is the signal sequence domain of CD8 alpha (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains from human CD8 alpha (1292-1507 nucleotides, GenBank NM 001768.6); An intracellular signaling domain from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-OX40Z CAR (2nd generation) is the signal sequence domain of CD8 alpha (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains from human CD8 alpha (1292-1507 nucleotides, GenBank NM 001768.6); An intracellular signaling domain from CD134 (733-840 nucleotides, GenBank AB590584.1); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-ZOX40L CAR (2nd generation) is the signal sequence domain of CD8 alpha (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains from human CD8 alpha (1292-1507 nucleotides, GenBank NM 001768.6); The intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and the intracellular signaling domain (141-206 nucleotides, GenBank NM 003326.4) derived from CD252 are linked to the CD3 ⁇ stop codon TGA.
  • NKG2D-28Z CAR (second generation) is the signal sequence domain of CD8 alpha (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge from human CD8 alpha (1292-1435 nucleotides, GenBank NM 001768.6); The transmembrane and intracellular signaling domains from CD28 (679-882 nucleotides, GenBank NM 006139.3); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-28 (H) Z CAR (2nd generation) is the signal sequence domain of CD8 alpha (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); CD28 derived hinge, transmembrane and intracellular signaling domains (562-882 nucleotides, GenBank NM 006139.3); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-BBOX40Z CAR (3rd generation) is the signal sequence domain of CD8a (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains from human CD8 alpha (1292-1507 nucleotides, GenBank NM 001768.6); An intracellular signaling domain from CD137 (901-1026 nucleotides, GenBank NM 001561.5); An intracellular signaling domain from CD134 (733-840 nucleotides, GenBank AB590584.1); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-BBOX40LZ CAR (3rd generation) is the signal sequence domain of CD8 alpha (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains from human CD8 alpha (1292-1507 nucleotides, GenBank NM 001768.6); An intracellular signaling domain from CD137 (901-1026 nucleotides, GenBank NM 001561.5); An intracellular signaling domain from CD252 (141-206 nucleotides, GenBank NM 003326.4); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-OX40BBZ CAR (3rd generation) is the signal sequence domain of CD8a (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains from human CD8 alpha (1292-1507 nucleotides, GenBank NM 001768.6); An intracellular signaling domain from CD134 (733-840 nucleotides, GenBank AB590584.1); An intracellular signaling domain from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-28BBZ CAR (Generation 3) is the signal sequence domain of CD8 alpha (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge domains from CD8 alpha (1292-1435 nucleotides, GenBank NM 001768.6); The transmembrane and intracellular signaling domains from CD28 (679-882 nucleotides, GenBank NM 006139.3); An intracellular signaling domain from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-28OX40Z CAR (3rd generation) is the signal sequence domain of CD8 alpha (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge domains from CD8 alpha (1292-1435 nucleotides, GenBank NM 001768.6); The transmembrane and intracellular signaling domains from CD28 (679-882 nucleotides, GenBank NM 006139.3); An intracellular signaling domain from CD134 (733-840 nucleotides, GenBank AB590584.1); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-28OX40LZ CAR (3rd generation) is the signal sequence domain of CD8a (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge domains from CD8 alpha (1292-1435 nucleotides, GenBank NM 001768.6); The transmembrane and intracellular signaling domains from CD28 (679-882 nucleotides, GenBank NM 006139.3); An intracellular signaling domain from CD252 (141-206 nucleotides, GenBank NM 003326.4); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-28 (H) BBZ CAR (3rd generation) is the signal sequence domain of CD8 alpha (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); CD28 derived hinge, transmembrane and intracellular signaling domains (562-882 nucleotides, GenBank NM 006139.3); An intracellular signaling domain from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-28 (H) OX40Z CAR (3rd generation) is the signal sequence domain of CD8a (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); CD28 derived hinge, transmembrane and intracellular signaling domains (562-882 nucleotides, GenBank NM 006139.3); An intracellular signaling domain from CD134 (733-840 nucleotides, GenBank AB590584.1); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-28 (H) OX40LZ CAR (3rd generation) is the signal sequence domain of CD8a (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); CD28 derived hinge, transmembrane and intracellular signaling domains (562-882 nucleotides, GenBank NM 006139.3); An intracellular signaling domain from CD252 (141-206 nucleotides, GenBank NM 003326.4); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • NKG2D-AAA-28 (H) OX40LZ CAR (3rd generation) is the signal sequence domain of CD8a (890-952 nucleotides, GenBank NM 001768.6); The extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); AAA (Triple field); CD28 derived hinge, transmembrane and intracellular signaling domains (562-882 nucleotides, GenBank NM 006139.3); An intracellular signaling domain from CD252 (141-206 nucleotides, GenBank NM 003326.4); And an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ and stop codon TGA.
  • VSVG-pseudotyped lentivirus 293T cells cultured in DMEM medium were incubated with various types of PCDH1-MSCV-CD16-construct-EF1-copGFP vector, EF1a-NKG2D construct vector or PCDH1-MSCV-EF1 -copGFP control vector, EF1a-GFP control vector (for producing mock-infected virus using a blank vector); And the HIV-based pPACKH1 lentivirus package kit (System Biosciences), using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif.).
  • CD16V constructs are: CD16V-ZO CAR, CD16V-BBOX40L CAR, CD16V-OX40L CAR, CD16V-ZOX40L, CD16V-Z CAR, CD16V- OX40BBZ CAR, CD16V-28BBZ CAR, CD16V-28OX40Z CAR, CD16V-28OX40LZ CAR, CD16V-28 (H) BBZ CAR, CD16V-28 (H) OX40Z CAR, CD16V-28 (H) OX40LZ CAR.
  • NKG2D constructs are: NKG2D-ZO CAR, NKG2D-BBOX40LZ CAR, NKG2D-BBOX40LZ CAR, NKG2D-OX40BZ CAR, NKG2D-ZOX40L CAR, NKG2D-ZZ CAR, NKG2D- (H) OX40LZ, NKG2D-28 (H) OX40LZ, NKG2D-28 (H) OX40LZ CAR, NKG2D-28OX40LZ CAR, NKG2D-28OX40LZ CAR, NKG2D-28OX40LZ CAR, NKG2D- .
  • Lentiviruses were prepared by adding various types of CD16V construct expression vectors or control plasmids to HEK293T cells densely packed in 80% flasks; And pPACKH1 lentivirus packaging plasmids. After 6 hours, the medium was replaced with DMEM medium containing 10% FBS. The conditioned medium containing lentivirus was collected 48 hours after transfection and filtered using a 0.45 ⁇ m filter unit (Milliopore, Billerica, MA, USA) to remove cell debris. The virus-containing viral supernatant was concentrated approximately 50-fold by centrifugation at 3000 rpm and 4 ° C for 20 minutes using an Amicon Filter (Millipore). The concentrated virus was stored at -80 ° C.
  • NK92MI cells in logarithmic growth phase were adjusted to a concentration of 1 x 10 6 cells / ml using Cellgro (Cellgenix) containing 1% human plasma, after which the lentivirus supernatant was diluted to 50-100 MOI Was added in the presence of 8 ⁇ g / ml polybrene and centrifuged at 1800 g for 90 minutes. After centrifugation, the cells were left in a humidified incubator at 37 [deg.] C and 5% CO2 for 48 hours. Cells were then washed twice with RPMI-1640 and then placed in RPMI-1640 containing 10% FBS until further use. Control cells were transduced using only the vector.
  • CD16V or NKG2D Detection of receptor expression including
  • CD16V CAR-transduced NK92MI cells NKG2D CAR-transduced NK92MI cells, control vector-transduced NK92MI (NK92MI-Mock), or NK-92MI cells were washed twice using FACS buffer, -AAD (Beckman coulter), anti-CD3, anti-CD56 and anti-CD16 (BD Biosciences) mAbs. Expression rates and mean fluorescence intensity (MFI) of stained cells were measured using BD LSRFortessa.
  • Transfection efficiency using NKG2D constructs was measured by flow cytometry analysis of cells expressing NKG2D among CD3-CD56 + cells.
  • NK92MI cells were first gated against a singlet and then gated against 7AAD- and CD3-CD56 +.
  • Transfection efficiency using CD16 constructs was measured by flow cytometry analysis of cells expressing GFP and CD16 among CD3-CD56 + cells.
  • Target cells were labeled with 30 ⁇ M Calcein-acetoxymethyl ester (Calcein-AM; Molecular Probes) at 37 ° C for 1 hour. After washing, the labeled target cells were distributed in 96-well plates at 1 x 104 cells per well. NK92MI cells were harvested, washed and then added at various E / T (effector-to-target) ratios and conditions with or without varying concentrations of rituximab. As a control, rituximab-independent anti-human antibody (Sigma aldrich) was used.
  • Example 2 containing an OX40 ligand (CD252) chimera CD20-positive < / RTI > of NK92MI cells expressing antigen receptor (CAR) Limpoma Assessment of cytotoxicity to cells
  • FCRG3A The V158 polymorphism of FCRG3A (CD16) is a high affinity immunoglobulin Fc receptor and is thought to have a beneficial effect in the treatment of antibodies.
  • V158 variant of FCRG3A (CD16) was prepared to generate the hinge and transmembrane domains of CD8a; T cell stimulating molecule CD3; And intracellular domains of various costimulatory molecules including CD28, 4-1BB, OX-40, and OX-40 ligands were combined in various combinations (Table 1).
  • CD16V-28Z CAR (second generation), CD16V-BBZ CAR (second generation), CD16V-OX40Z CAR (second generation), or CD16V-28OX40LZ CAR (3rd generation)) was expressed in NK92MI cells using a lentiviral vector containing the MSCV promoter. Whether respective CARs are expressed on the surface of NK92MI cells was confirmed by detecting human CD16 using monoclonal mouse anti-human antibodies. Repeated experiments using flow cytometry showed that CARs were transfected with 90% or more efficiency in NK92MI cells (FIG. 1A), and the amount of lentiviral vector used was 50 or more (MOI, multiple of infection) .
  • CD252 OX40 ligand
  • Combined CD16V Receptor NK92MI Increase of apoptosis in CD20-positive rimforma of cells
  • CD16V-ZZ CAR first generation
  • CD16V-28Z CAR second generation
  • CD16V-BBZ second generation
  • CD16V-BBZ second generation
  • CD16V-OX40Z CAR second generation
  • CD16V-28OX40LZ CAR third generation
  • cytotoxicity of NK92MI cells expressing CARs according to the present invention to K562 was similar to that of K562 of the control (Mock) transduced with the vector alone. This means that the cytotoxic ability of NK92MI cells to the target was not increased by side effects due to the genetic modification itself (FIG. 1B).
  • 5: 1, 2.5: 1, 1: 1 and 0.5: 1 represent the ratio of the number of NK92MI cells as effector cells to the number of K562 cells as target cells.
  • NK92MI cells expressing CD16V-containing receptors transfected NK92MI cells and cancer cells (B-cell lymphoma cell line Ramos) were co-cultured and the lysis of cancer cells was evaluated using a Calcein-AM release assay (Fig. 1C).
  • Fig. 1C 5: 1, 2.5: 1, 1: 1 and 0.5: 1 represent the ratio of the number of NK92MI cells as effector cells to the number of target cells Ramos cells. Since NK92MI cells themselves do not express CD16, it is known that NK92MI-induced apoptosis does not increase even in the presence of rituximab.
  • CD16V-ZZ CAR first generation
  • CD16V-28Z CAR second generation
  • CD16V-BBZ CAR second generation
  • CD16V-OX40Z CAR NK-92MI cells expressing the CD16V-28OX40LZ CAR (3rd generation) or the CD16V-28OX40LZ CAR (3rd generation) were found to have strong killability on Ramos cells at 5: 1 and 2.5: 1 working cell: target cell ratio in the presence of rituximab Respectively.
  • CD16V-28OX40LZ CAR which is a third generation CAR including OX40 ligand
  • CD16V-OX40LZ CAR was prepared to evaluate whether the second generation CAR containing OX40 ligand could enhance the anticancer activity of NK92MI cells. No expression was observed when CD16V-OX40LZ was transfected into NK92MI cells using the lentiviral vector (Fig. 1D) and did not show the ability to kill Ramos cells in the presence of rituximab (Fig. 1E). Therefore, the present inventors focused on the fact that the OX40 ligand is a type II protein and that the CD3 ⁇ ligated to the OX40 ligand is a type I protein.
  • CD3 ⁇ is attached to the N-terminus and OX40 ligand is replaced with CD16V-ZOX40L CAR Respectively.
  • the lentiviral vector containing the MSCV promoter was used to transfect NK92MI cells with CD16V-ZOX40L, it was confirmed that they were effectively expressed differently from CD16V-OX40LZ (Fig. 1F).
  • the anticancer activity of the second generation CAR in which CD16V-ZOX40L was introduced was evaluated, it showed strong killing ability against Ramos cells at a ratio of 10: 1, 5: 1 and 2.5: 1 working cells: target cells in the presence of rituximab (Fig. 1G).
  • CD16V-28OX40LZ CAR (3rd generation) containing OX40 ligand CD16V-ZOX40L CAR (2nd generation) was also found to be superior to CD16V-Z CAR, a positive control, in anticancer activity.
  • OX40 ligands were introduced into CARs based on the CD137 (41BB) signaling domain to assess expression and cytotoxicity in NK92MI cells. Both CD16V-BBZ CAR (second generation) and CD16V-BBOX40LZ CAR (third generation) were expressed at high levels in NK92MI cells (FIG. 2A). CD16V-Z, CD16V-BBZ, and CD16V-BBOX40LZ showed low killing activity against Ramos cells when there was no rituximab in the in vitro cytotoxicity test, but when rituximab was present, all the working cells: target cells CD16V-BBOX40LZ in combination with OX40 ligand showed strong cytotoxicity (Fig. 2B).
  • the present inventors compared the cytotoxicity of NK92MI cells expressing CD16V-28OX40LZ CAR and NK92MI cells expressing third generation CAR having different intracellular signaling domains. It was confirmed that the third generation CARs (CD16V-28OX40LZ CAR, CD16V-28OX40Z CAR, and CD16V-28BBZ CAR) used in this experiment were all expressed at high levels in NK92MI cells (FIG. 3A).
  • FIG. 3B shows the results of comparing the NK cell activation effect of various third generation CARs according to the present invention.
  • all of the above CARs showed low cytotoxicity against Ramos cells when there was no rituximab and similar degree of cytotoxicity.
  • CD16V-28OX40LZ CAR, CD16V-28OX40Z CAR, and CD16V-28BBZ CAR showed high cytotoxicity against Ramos cells.
  • CD16V-28OX40LZ CAR including OX40 ligand showed the best cell killing ability (FIG. 3B).
  • hinge sequence and composition between antigen-specific receptor and cell membrane are important as well as identification of cancer cell antigen recognition receptor.
  • the sequence and configuration of the hinge may need to be designed differently depending on the target molecule.
  • the CARs used in the experiments described above all used CD8 ⁇ fragments as spacer hinges.
  • CD28 fragments in the hinge to evaluate how the cell killing capacity of these CARs change is summarized in FIG.
  • FIG. We have constructed a lentiviral vector containing the extracellular domain of CD16V (ectodomain) and the hinge of CD28.
  • the hinge, transmembrane and intracellular signaling domains derived from CD28 were connected to signaling modules of CD134 (OX40), CD137 (4-1BB), or OX40 ligand (CD252) .
  • the third generation CARs thus produced were expressed in NK92MI cells using lentiviral vectors.
  • the transfected NK92MI cells were found to express high levels of the respective third generation CAR CD16V-28 (H) BBZ CAR, CD16V-28 (H) OX40Z CAR or CD16V-28 (H) OX40LZ CAR 4A).
  • CD16V-28 (H) BBZ CAR, CD16V-28 (H) OX40Z CAR and CD16V-28 (H) OX40LZ CAR showed cytotoxicity when there was no rituximab
  • CD16V-28 (H) BBZ CAR, CD16V-28 (H) OX40Z CAR and CD16V-28 (H) OX40LZ CAR showed strong cytotoxicity when simps were present.
  • CD16V-28 (H) OX40LZ CAR containing OX40 ligand exhibited the highest cytotoxicity.
  • CAR which includes the novel CAR, especially OX40 ligand, discovered by the present inventors as an intracellular signal transduction domain, exhibits excellent anticancer effects when expressed in natural killer cells.
  • Example 3 containing an OX40 ligand (CD252) NKG2D chimera Antigen receptor ( NKG2D -CAR) < / RTI > NK92MI Evaluation of cytotoxicity of human breast cancer cells and lung cancer cells
  • NKG2D Receptor NK92MI Cell MCF7 Breast cancer Increased cell death to cell line effect
  • cytotoxicity of NK-92MI cells expressing NKG2D CAR to MCF7 breast cancer cells was compared by Calcein-AM release assay.
  • NK-92MI cells expressing NKG2D CAR were cultured and analyzed by Calcein-AM release Tumor cell lysis was measured.
  • Previous studies have shown that the addition of ancillary molecules to chimeric receptors increases the cytotoxicity of T and NK lymphocytes.
  • NKG2D CAR we introduced the signal transduction domain of CD28, CD134 (OX-40) or CD137 (4-1BB), the best known three co-stimulatory molecules into NKG2D CAR.
  • Third generation CAR is known to increase antitumor activity.
  • optimal lymphocyte activation requires one or more co-stimulatory receptors coupled with co-stimulatory molecules such as CD28.
  • co-stimulatory molecules such as CD28.
  • the most important accessory stimulating receptors are CD137 (4-1BB) and OX40 (CD134), which are members of the tumor necrosis factor (TNFR).
  • NKG2D CAR (third generation) constructs were expressed in NK92MI cells using lentiviral vectors.
  • Transfected NK92MI cells efficiently expressed various NKG2Ds containing third generation CAR (FIG. 6A).
  • NK92MI cells expressing various NKG2D CARs (3rd generation) effectively lysed MCF7 cells in Invitro.
  • NKG2D-28OX40Z CAR and NKG2D-28BBZ CAR containing CD134 or CD137 co-stimulatory domains in the framework of NKG2D-28Z CAR did not show higher cytotoxicity than NKG2D-Z CAR (first generation) 6B).
  • NKG2D-28 (H) OX40LZ CAR 3rd generation
  • AAA triple alanine linker between the extracellular domain of NKG2D and the CD28 hinge.
  • NKG2D-AAA-28 (H) OX40LZ CAR (third generation) with AAA linker was expressed in NK92MI cells using lentiviral vectors (Fig. 7A).
  • NKG2D-AAA-28 (H) NKG2D ligand positive tumor cells were transfected with NKG2D-AAA (SEQ ID NO: 2) to evaluate the ability of NK92MI cells transfected with OX40LZ CAR (3rd generation) to recognize NKG2D ligands in lung cancer cells -28 (H) OX40L CAR (3rd generation).
  • Transfected NK92MI cells efficiently expressed NKG2D-Z CAR (first generation) or NKG2D-AAA-28 (H) OX40L CAR (third generation) (Fig. 8A).
  • expression of NKG2D ligand in H1299 and H1944 cells was evaluated.
  • NK92MI cells containing NKG2D-AAA-28 (H) OX40L CAR (3rd generation) were more effective than control NK92MI cells and NKG2D-Z CAR (first generation) expressing NK92MI cells in the presence of NKG2D ligand- Cells could be dissolved more efficiently.
  • Specificity was evident through the absence of dissolution of H1299 and H1944 cells by NK92MI cells expressing the control vector that did not express NKG2D.

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Abstract

La présente invention concerne un récepteur antigénique chimérique et des cellules tueuses naturelles exprimant celui-ci et, plus particulièrement, un récepteur antigénique chimérique (CAR) qui comprend un domaine de signalisation intracellulaire comprenant tout ou partie d'un ligand OX40 (CD252), ayant ainsi un excellent effet d'amélioration de l'activité anticancéreuse sur des cellules immunitaires, et des cellules immunitaires exprimant celui-ci.
PCT/KR2017/015635 2016-12-28 2017-12-28 Récepteur antigénique chimérique et cellules tueuses naturelles exprimant celui-ci WO2018124766A2 (fr)

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CN110121505A (zh) 2019-08-13
JP2020513770A (ja) 2020-05-21
AU2017384900A1 (en) 2019-06-20
JP6971319B2 (ja) 2021-11-24
US20190336533A1 (en) 2019-11-07
WO2018124766A2 (fr) 2018-07-05
CN110121505B (zh) 2023-08-01
EP3567049A2 (fr) 2019-11-13
EP3567049A4 (fr) 2020-08-26
US20230025506A1 (en) 2023-01-26
CA3061898A1 (fr) 2019-10-29
WO2018124766A3 (fr) 2018-12-13

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